EP1885846A2 - Epigenetic and genetic treatment method and system - Google Patents

Abstract

The invention relates to an epigenetic and genetic treatment method and system. The invention can be used to rejuvenate an adult cell nucleus without passing through an embryonic stage and to obtain a tissue that is genetically-rejuvenated and autologous in relation to the original nucleus, comprising the temporary introduction of a nucleus into an enucleated oocyte (genetic rejuvenation), followed by oocyte extraction prior to cell division. The treated nucleus can then be introduced into an adult cytoplasm originating from the nucleus. In this way, cell divisions from the rejuvenated nucleus take place in an adult autologous cytoplasm and the resulting cells immediately take the form of adult, differentiated and functional cells. The rejuvenated autologous tissue thus created can then be grafted without rejection to the adult tissue of the organism from the treated nucleus. Throughout the inventive procedure, the treated cytoplasms and nuclei remain permanently in the adult state and no artificial cell differentiation is necessary. In this way, the invention is different from standard deep cloning in which the treated cells must return to the embryonic stage and which involves artificial cell differentiation. In addition, the tissues derived from standard cloning are not immunologically compatible with the original adult tissues from the treated nucleus since the cell division thereof takes place in the oocyte which represents a foreign cytoplasm to the nucleus. Moreover, standard cloning enables the creation of whole organisms unlike the inventive method and system which can only be used to treat a single tissue. The possible applications of the invention include, for example, the repair of necrotic tissues, such as a myocardial infarction, and damaged tissues, such as the retina (RMD), heart failure, renal failure, liver failure and osteoporosis, the treatment of cancers that occur with age, such as prostate cancer, rectal cancer, colon cancer, etc. The anodyne implantation of a small volume of autologous, rejuvenated, still-healthy cells from the affected organ should inhibit the aforementioned cancers and the metastases thereof.

Description

 System and method for genetic and epigenetic treatment

The present invention provides systems and methods for genetically and epigenetically treating cells.

Such systems and methods are particularly useful in the field of cellular treatments, in particular for producing autografts from differentiated cells or embryonic or fetal strains.

Cellular treatments, and in particular autografts, are currently practiced to repair damaged tissue suffering from a disease, cell deficit or necrosis. This technique generally involves taking a few healthy cells of the tissue concerned, to cell culture these cells to form a cell stock or tissue and to re-implant these cells in the tissue to be treated. These reprogrammed cells can then allow the tissue concerned to recover its original morphological and functional capabilities.

For example, this technique is used for the repair of articular cartilage. Joint cartilage has limited repair potential and lesions of a certain volume rarely heal well. In order to repair such lesions and prevent the appearance of osteoarthritis in patients, chondrocytes, bathed in an extracellular matrix, are removed, removed from the matrix, for example by enzymatic digestion and then cultured, generally on sera of fetal calf or preferably in the patient's serum, and in three-dimensional matrices (for example an agarose, collagen or globin matrix). Such cultivation then allows the collected cells to multiply by mitotic division and then results in the production of millions of chondrocytes. These chondrocytes can then be reimplanted into the cartilaginous tissue to restore deficient cells and cartilage.

However, the disadvantage of these multiplication techniques is that generally the cells removed are cells that have already undergone numerous mitotic divisions. However, cell culture for their multiplication causes a slight decrease in telomeres at each mitosis and this multiplication is often done on cells already aged, close to their end of life and their end of functions, and whose DNA is in besides likely to be altered. In particular, it is known that cellular aging results in a gradual gnawing of telomeres (end of chromosomes). Now these telomeres condition the remaining number of mitotic divisions. Thus, culturing mother cells that have already undergone numerous mitoses may result in a large colony of aged daughter cells with shortened survival that may also exhibit alterations in gene functionality.

The object of the present invention is to provide systems and methods for genetic and epigenetic treatment overcoming the aforementioned drawbacks.

In particular, the present invention aims to provide systems and methods of cell processing for rapid mass production of healthy cells with improved gene functionality and / or capable of being genetically and epigenetically rejuvenated, aged and / or repaired to a desired degree.

Still another object of the present invention is to provide systems and methods for cell processing resulting in both reconstitution of missing, failing, enhancing or modifying autologous tissue, and genetic rejuvenation of tissue in which cells have been implanted. The subject of the present invention is therefore a system for the genetic and epigenetic treatment of cells to be treated, comprising: at least one cell to be treated,

a genetic reprogramming medium (MRG), comprising at least the natural cytoplasm of at least one genetic reprogramming cell (CRG) and / or the synthetic cytoplasm, and means for contacting at least a portion of at least one nucleus of at least one cell to be treated with said MRG to modify the biological age and / or repair said at least one cell to be treated.

The subject of the present invention is also a method of genetic and epigenetic treatment of cells to be treated, comprising the following steps:

provide at least one cell to be treated,

- provide a genetic reprogramming medium (MRG), comprising at least the natural cytoplasm of at least one genetic reprogramming cell (CRG) and / or synthetic cytoplasm, and - contacting at least a portion of at least one nucleus of at least one cell to be treated with said MRG to modify the biological age and / or repair said at least one cell to be treated.

Various embodiments and applications are described in the dependent claims. Advantages, features and applications of the invention will emerge more clearly in the following detailed description of several embodiments and variants of the invention.

The invention relates in particular to the modification of the environment of a cell nucleus with or without extracellular or intra-oocyte transfer, so as to put the nucleus under the influence of a medium inducing its partial genetic reprogramming, but without causing a return of the nucleus to the development of embryonic cells. This medium will cause better repair of cellular DNA during division and aggression and / or genetic rejuvenation by the action of an inverting medium of biological time, such as an oocyte.

The present invention relates in particular to systems and methods applicable to the field of treatment and / or repair and / or functional and / or morphological cellular enhancement intended to open many perspectives for the fight against a large number of diseases but also against Tissue senescence is largely due to the loss of their functional and morphological capacity for proliferation, regeneration and repair. In particular, the present invention relates to a system and methods capable of treating cells of a tissue, in particular for rejuvenating, aging and / or repairing these cells. The cells are then cultured in a suitable medium so as to create a stock or tissue of genetically and epigenetically treated cells capable of being implanted in the tissue in question, or at a distance from it, where these cells can emit particular proteins and and / or signaling peptides and / or stimulation of metabolism, and / or DNA repair enzymes, for the tissue in question.

More specifically, the systems and methods of the invention comprise contacting at least a portion of a nucleus of at least one cell to be treated with a genetic reprogramming medium (MRG).

This MRG comprises at least the natural cytoplasm of at least one genetic reprogramming cell (CRG) and / or synthetic cytoplasm. A reconstituted and / or synthetic cytoplasm can be composed in particular of embryonic serum extracts, cicatrization and / or cells subjected to metabolic activation. A completely synthetic cytoplasm, for example made by physical and chemical reconstitution of active substances, is possible. It is also conceivable to produce a MRG in the form of a "slurry" of CRG, with or without cores. It is also possible to add cell extracts or cytoplasm and / or other substances known to be capable of activating nuclear metabolism, such as cells or cell extracts appearing during healing and / or signaling or metabolism-stimulating proteins or peptides and / or growth factors and / or cells or extracts of cancer cells.

The cell or cytoplasmic extracts can be obtained by well-known physical or chemical treatments. The advantage of using cancer cells, and in particular the cytoplasm thereof, is explained by the fact that metabolic activation, signaling and mitosis factors are particularly intense and may induce a temporary metabolic or nuclear reactivation of a cell to be treated. Cancer contagion is unlikely because cancers are usually not transmissible to a different tissue, and their cytoplasm remains normal. The use of cytoplasm of selected cancer cells is possible to temporarily treat nuclei or cells, adult or otherwise, that are insufficiently capable of dividing spontaneously or in culture, to repair their poorly copied DNA, or which are functionally deficient. It is known that a cancer cell rapidly enlarges its telomeres, accelerates and prolongs its mitosis indefinitely, increases its DNA repair enzymes and increases its self-para- and endocrine performance. It is therefore a matter of selectively transferring a selected operation of the cancer cell to the cell to be treated, without risking a teratogenic neoplastic contamination. Since cancer is not directly contagious for non-cancer cells, even if they are ill, the selective regenerative application of cancer cells can be done, for example, in two ways: intracellular treatment: a nucleus is taken from a cancer cell and introduced into the cytoplasm of a tissue cell to treat; this nucleus preferably remains separated from the normal nucleus by a tongue or membrane of a porous biocompatible fabric possibly impregnated with antibodies, said tissue permitting the signaling proteins but preventing any passage of genes or chromosomes. Under these conditions, the signaling proteins emitted by the cancerous nucleus will cause a partial genetic reprogramming of the cell nucleus to be treated, especially in its defective functions. After one or more mitoses, the cancerous nucleus will be removed, and the reprogrammed cell may be multiplied in a suitable cell culture. extracellular treatment: selected cancer cells are approximated with a few cells to be treated in a cell culture bath adapted to include at least one factor capable of increasing the permeability of cell membranes, particularly to signaling proteins. This can be done until the observation or genetic, proteomic, biochemical or biophysical tests allow to note the induction of a functional reactivation of the nuclear deficiency (s) to be corrected. In particular, selective biochips will make it possible to target the signaling proteins emitted from the cancerous nucleus and selectively reprogram the nucleus to be treated in its intended and programmed functions. Three main types of treatment can be considered, namely the rejuvenation of the biological age of a cell, the aging of the biological age of a cell and the repair of a cell. Rejuvenation of the biological age of a cell also promotes self-repair capacity of this cell, especially at the level of its DNA.

As part of a genetic and epigenetic treatment to rejuvenate a cell, MRG includes all or part of one or more CRGs. In this case, such a CRG is advantageously an oocyte, an embryonic cell, an embryonic or adult stem cell, a fetal cell, or a cellular container reconstituted from these cells, or synthesized. The systems, methods and applications for achieving such rejuvenation will be described in more detail below. The aging of a cell is particularly conceivable for treating fetal diseases or in the newborn, due in particular to cancers of embryonic origin, such as glioblastoma. To reprogram these cancerous cells, they can be artificially aged by replacing the cancerous nucleus with a healthy nucleus of the same autologous or homologous but older tissue, preferably HLA (Human Lymphocyte Antigen) compatible. Thus, the interaction between the older nucleus and the young cytoplasm promotes some aging temporarily accelerated at least the cytoplasm of the young cell. Aging can then be increased by multiplication of cells in a culture bath, the young cytoplasm causing accelerated mitoses of the older nucleus, inducing a decrease in telomeres. After culturing, healthy cell sorting is possible to re-implant healthy tissue in place of the original diseased tissue.

Another possible application is to repair a cell, in particular in its chromosomal constitution, by treating only a part of a nucleus, for example a chromosome. For example in the context of leukemia, the chromosome, particularly the "Philadelphia" chromosome, may be destroyed during the metaphase where the chromosomes are deployed, for example by means of an ultrathin laser beam, preferably diameter equal to or less than 1 micron. Then we take a healthy equivalent chromosome in the metaphase of an equivalent cell of the patient or a compatible HLA donor, and is implanted in the cancer cell, especially during its mitosis. The treatment could be considered in particular for a large number of cancers, for example glioblastoma, cancer of the breast or rectum.

Another possible application is to repair only part of the chromosome. Thus, one can remove a specific part of a chromosome, for example the part whose genes are responsible for graft rejection. This can be done using an ultrathin laser beam. Then we take the equivalent part of the equivalent chromosome in the recipient of the graft, which can also be done by laser cutting using an ultrathin laser beam. This part of the chromosome is then reintroduced into the original chromosome, which can be done using plasmids, phagemids, synthetic vectors, or micromanipulations in nanotechnology. Synthetic vectors are made in the laboratory and consist of structures called "copolymer blocks" that bind to DNA or RNA. More generally, this type of repair is conceivable to repair any deficiency or dysfunction of a cell part, especially due to age. Various embodiments and applications of cellular rejuvenation will now be described in more detail.

According to a first aspect of the invention, in order to rejuvenate or regenerate a differentiated cell, its nucleus (with or without its attached cytoplasm) is removed and transferred to the MRG, advantageously in a CRG of the oocyte, embryonic, fetal cell or cancerous. This core is left in the MRG for a predetermined time and then removed. According to a first variant, the nucleus is removed before the end of the nucleus telophase, that is to say that the nucleus is extracted from the MRG before its division into two cells, that is to say before the end of its first mitosis. The inventor It has been observed that this temporary introduction of a nucleus into an MRG, especially in a CRG, results in a rapid and important lengthening of the telomeres, often synonymous with rejuvenation of the chromosomal material. The regenerated nucleus can then be introduced into a differentiated recipient cell, strain or embryo, preferably enucleated, preferably autologous with respect to said nucleus, preferably of identical tissue, advantageously in the cell of origin of the nucleus or a neighboring cell, in which mitotic division can continue and thus lead to the birth of two daughter cells whose nucleic material is regenerated. Then, these cells may be subjected to a multiplication culture and reach such quantities that at least millions of cells sufficiently differentiated to be functionally and morphologically able to be implanted in the original tissue concerned. Alternatively, the nucleus can be removed from the MRG after one or more mitoses, then one (or more) of the rejuvenated nuclei thus obtained is (are) reintroduced into a differentiated receptor cell, advantageously autologous with respect to the nucleus. preferably in the cell of origin of the nucleus or a neighboring cell.

It should be noted that in the context of this first aspect of the invention, it may be desirable to open or at least partially remove the CRG membrane, to avoid any risk of cell division thereof. Indeed, the membrane is necessary for the cell division phenomenon, whereas it is the cytoplasm that is the privileged place of genetic reprogramming. According to an advantageous aspect of the invention, the step of collecting and transferring the nucleus of the differentiated cell comprises taking, in addition to the nucleus, at least a part of the cytoplasm contained in the differentiated cell, in order to find in the MRG, particularly in CRG, some cytoplasmic components initially present in the cell differentiated, such as endoplasmic reticulum, golgy apparatus, ribosomes and / or mitochondria.

According to a second aspect of the invention, the bringing into contact of at least the nucleus of a differentiated cell with said MRG may consist of transferring MRG into a differentiated cell, for example by means of a pipette or by induced transfer. by a pressure difference. This can be accomplished by creating at least one slit or aperture in the membrane of the differentiated cell, and transferring MRG into said differentiated cell through said at least one slit or aperture. Advantageously, provision is made to be able to separate or remove said transferred MRG after a certain predetermined or observable time, sufficient to genetically reprogram the nucleus of the differentiated cell. For example, it is conceivable to have a CRG and a differentiated cell side by side, to make an opening in the CRG membrane and an opening in the membrane of the differentiated cell, then to compress the CRG to at least partially transfer the cytoplasm of CRG in the differentiated cell. This compression can be obtained by placing a pipette or the like above the membrane of the cell to be compressed, preferably locked against a wall, and exerting a suitable pressure. This pressure could also be exerted by means of a fluid, preferably viscous, which can overflow the pipette without detaching it. This compression is maintained for the time necessary for the genetic reprogramming of the nucleus of the differentiated cell, then the compression on the CRG is suppressed, with the effect that the cytoplasm of the CRG transferred into the differentiated cell is at least partially re-aspirated into the cell. MAF. It should be noted that MRG can be removed before or after the first mitosis of the differentiated cell nucleus. Alternatively, means may be provided for closing the differentiated cell with at least a portion of the remaining MRG included therein. The examples of applications described hereinafter more generally refer to the first aspect of the invention described above (temporary transfer of a differentiated cell nucleus in an MRG, in particular in a CRG), but it is understood that they could all be implemented also with the second aspect of the invention described above (transfer of MRG in a differentiated cell). On the other hand, most of the examples refer to oocyte use, but any CRG, and more generally any MRG can be used to implement these examples.

First, it should be noted that the oocyte used may possibly be a mammalian oocyte. For example, a rabbit or sheep oocyte could be used. Oocytes derived from differentiation induced from embryonic stem cells (OPCE) can also be created in vitro. These OPCEs, obtained for example by cloning, can come from the recipient of the grafts and the treated nuclei thus become particularly autologous because the cytoplasm of the OPCs will comprise only a part of its foreign DNA and / or RNA, especially in the mitochondria and ribosomes. It is also possible to introduce into the oocyte, a nucleus, which is, for example, in early, spontaneous or induced mitosis, or else chromosomes or genes or parts of nuclei to be treated in an embryonic-type cell. Embryo-like cells that may be artificially activated by proteins or peptides of genetic signaling or cell activation or regulation may also be used, providing an environment capable of inducing some neighborhood genetic reprogramming. Thus, removal of the nucleus from the differentiated cell can be advantageously done in anaphase or during telophase depending on the degree of genetic rejuvenation desired. In order to observe the current mitotic period, optical means, such as a microscope, may be used. If a CRG is then used, it will then preferably come from the same tissue, for example, cartilage, myocardial, etc., preferably partially pitted and cultivable in vitro, in vivo or in situ. This or these cell (s) will be cultured for propagation preferably a sufficient time in vivo in embryonic tissues to obtain a partial dedifferentiation. The nuclei thus treated can be left either in the embryonic-type cells to constitute a graftable tissue in the organism of origin of the nucleus, or extracted from their receptor cells to become inducer of local cellular or trans-membrane local regeneration at the level of the nucleus. a differentiated tissue, preferably autologous and identical. The implantation of the nucleus or nuclear part can also be carried out inside a stem cell, preferably of embryonic or fetal type.

Such partially and selectively dedifferentiated cells can then be introduced into differentiated cells, such as chondrocytes, immune function, endocrine, cardiac cells, tissue cells that have undergone anti-cancer treatment, β cells and α islets of Langerhans, cells of the same origin as a graft to be transplanted, hepatocytes, etc., in order to regenerate the corresponding tissue. Such an invention can thus be applied without limitation to the regeneration of any sufficiently differentiated cell, such as cardiac, renal, bone, tendinous, cartilaginous, cutaneous, dermal, epidermal, pancreatic, hepatic, nerve, prostatic, glandular or hematopoietic cells. nerve, vascular, retinal, dental, desmodontal, splenic, parathyroid, adrenal, digestive and respiratory tract, etc. From a certain degree of dedifferentiation, these cells lose their immunogenicity and can sometimes be used to regenerate non-autologous tissues. This function also comprises the ability of these genetically activated cells to act remotely by secretion, release or induction of peptides and / or genetic signaling proteins, in particular, by specific biochemical molecules. This trans-membrane and / or trans-humoral genetic activation gives these cells the ability to actively and continuously stimulate other senescent deficient cells or to inhibit carcinogenic factors. The system according to the invention and the cell regeneration methods used preferably comprise four successive stages, namely a preparation of the nuclear material, a genetic reprogramming, a multiplication in culture and a reimplantation in the organism of origin of the nucleus. The preparation of the nuclear material consists in taking the nucleus of the cell, preferably differentiated with more or less cytoplasm, in order to preserve, if possible, the cytoplasmic components, such as the mitochondria, the ribosomes, the endoplasmic reticulum, the Golgi apparatus, lysosomes, peroxisomes, etc., of the initial differentiated cell at the level of the oocyte hosting this sample. The inventor assumes that this step ensures synchronous reprogramming of the various vital structures around the nucleus and a probable conservation of the cellular "morpho-temporal field". Moreover, it is possible that certain constituents of the nuclear material such as chromosomes, a set of genes, one or more isolated genes (natural, recombined, semi-synthetic or synthetic) have previously been subjected to such a regeneration process. In this way, some elements of the preparation will have a different biological age. In addition, it may be provided to join a segment including plant DNA or synthetic vectors such as copolymer blocks, coding for example for vitamin C, E, folic acid, essential amino acids, unsaturated lipids essential peptides such as natriuretic (BNP) or atrial natriuretic (ANP) brain peptides, peptides C and Y, glutathione, peptide hormones such as glucagon, insulin, ACTH, peptides antibiotics, proteins such as globulins, immunoglobulins and albumins, DNA and RNA repair, restriction, replication and transcription enzymes, as well as cytochromes, cytokines, etc. to a gene or a chromosome, expressing for example erythropoietin or various albumin for example during metaphase, or the nuclear membrane to anaphase, telophase or a corresponding interphase. The inventor estimates that the increase in cell biological age results in a decrease in the high elasticity of chromosomes, in particular metaphase chromosomes. This, in turn, leads in particular to a decrease in the accessibility of DNA polymerases, endo- and exonucleases and chromosomal DNA enzymes and thus constitutes an epigenetic and genetic cause of aging which can be combated by the genetic rejuvenation of the genome. 'invention.

The membranocytoplasmic container (oocyte) for treating the cytoplasmic reprogramming elements may sometimes be too small for the cellular elements to be treated. By way of example, one can think of the simultaneous treatment of a nucleus with a part of its cytoplasm, or of several sometimes different nuclei such as in a nephron, a muscle cell, a myocardial autorythmic cell, a hair follicle, a unit epidermal melanization, an epidermal-dermal unit, a glandular unit, a hepatobiliary unit, a retinal functional unit (such as a pigmented epithelium: cones, rods, bipolar cells, horizontal cells and Mϋller cells), a vascular unit (cell endothelial and myoarterial), a hematopoietic unit, a neuro-glio-dendritic unit, an ovarian unit of Graaf's follicles, etc. In order to treat a multi-nucleus unit with a regeneration system or method according to the invention, it may be necessary to have an enlarged membranocytoplasmic vessel with preserved oocyte function (RAF). Such an RAF can be made by gluing the membranes respective mammalian autologous or autologous oocytes, for example by manual or robotic micromanipulations, preferably preserving each respective cytoplasm within their respective membrane and creating a spherical, ovoid or cylindrical type volume. Such manipulation requires preserving the vital environment for each oocyte. This membrane bonding may for example be achieved by means of a micro laser beam, a small heating light ray, a biological adhesive, etc.

The in vitro multiplication is preceded by the introduction of the nucleo-cytoplasmic material into a preferably enucleated cell, identical to that from which the nucleus originates, and with at least recoverable vitality. Current multiplication techniques allow, for example, in two weeks to obtain about half a billion cells from tens. In the present case, the inventor has observed that the regeneration method according to the invention ensures a rapid and significant increase in the length of the telomeres in less than a day, thus making it possible to counterbalance their shortening resulting from so many successive replications. This multiplication can also be done in vivo but is generally much slower and often requires sufficient initiation in vitro. This decreases the amount of cells required as well as the significant elongation of the telomeres and probably allows for better functional adaptation as well as greater genetic influence at a distance.

It may be desirable to regenerate together a plurality of cells representing an organic functional unit, such as for example a nephron, retinal pigment cells of different categories or pulmonary alveoli, to form a genetically rejuvenated organic functional unit. For example, a cellular micromanipulation can be envisaged to produce an enlarged enclosure with a genetic and epigenetic reprogramming function capable of reversing temporally the evolution of the biological age of the nuclei and / or multiple cytoplasm that are introduced there. For this purpose, cells with an oocyte function are, for example, cut into two parts, preferably by a cold-light laser micro-ray. Both of these portions are open, and their membranes may be attached to a protein layer, such as globin, which has preferably been applied to a flexible surface. This carpet of oocyte or embryonic membranes presents the cytoplasm upwards. When a sufficient surface of such a cytoplasmic velvet (VC) is formed, several differentiated cell nuclei can be placed there, with or without their cytoplasm, and then roll the VC around them as close as possible. This interactive cell sandwich will preferably remain in the conventional cell culture nutrient liquid for the desired time to achieve the desired mitosis phase. It is thus possible to obtain the simultaneous rejuvenation of several nuclei belonging to an organic functional unit which can be multiplied either in the state of isolated cells, which requires a rearrangement of the cells multiplied according to their functional order, or in the state of a set of cells already placed in their functional order.

The regeneration method according to the invention is particularly suitable for diseases characterized by a deficit or a cellular failure

(diabetes, myocardial infarction, hepatitis, renal failure, decreased retinal function, or genetic diseases responsible for immunological deficiency) as well as for cancers occurring after a certain age, such as that of the prostate, breast and of the colon. This cell regeneration is applicable to many cell types and can therefore create controlled regeneration tissues to treat many organic and tissue lesions. For example, by taking, under ultrasound control, a transrectal or transdermal needle, or an endoscopic prostates that will be totally or partially treated and by relocating them, for example, into the prostate, this remote inductive cellular rejuvenation, in particular by signaling proteins, may, in some cases, oppose the development of a local cancer, slow down its extension or even destroy all its metastases. Thus also, an autologous or even homologous ophthalmic retina, including a functional cellular unit, for example consisting of a few cells of the pigmented epithelium, cones, rods, bi-polar cells and / or Muller cells, which has taken and regenerated, may be of great interest in the case of AMD. Severe renal insufficiency can be combated by the implantation of partially dedifferentiated cells obtained, for example after transfer into and out of oocytes, of nuclei of different nephron cells. Osteoarthritis can be treated by implantation of chondrocytes from cell regeneration. The same applies to cutaneous surfaces and hair follicles, and in particular to regenerate and / or color bleached hair, for example by transferring into one or more oocytes (s) preferably one or more nuclei or parts of cell nuclei. hair follicle, melanocytes and keratinocytes, for regeneration of the hair and / or its color. Yet another application of the invention may consist in reinforcing or recreating the thymic functions by genetic rejuvenation of homologous or, if possible autologous, sufficiently dedifferentiated thymic cells to actively resuscitate the immunoprotective functions of the body.

Various applications of the system and method according to the invention will now be described in more detail. The set of steps necessary for cell regeneration will not be repeated thereafter, the overall principle remaining the same and can be adapted to each case by the skilled person. It is also recalled that both aspects of the invention, namely on the one hand the temporary transfer of a differentiated cell core in a MRG (in particular a CRG), and secondly the transfer of MRG in a differentiated cell, are usable.

Thus, we know that aging causes kidney failure with progressive anemia, both factors bringing great chronic fatigue in these people. Here, it is advisable to promote renal regeneration, in particular by reconstituting a few nephrons as well as cells producing erythropoietin.

A kidney can be regenerated in vitro from the various nephron cells (CNE) obtained for example by surgical or endoscopic renal biopsy under visual control. The CNEs will be treated by the treatment of the invention in order, for example, to reduce their biological age by three quarters, and the CNEs thus obtained will be amplified. Simultaneously, a whole block of nephrons (BNE) was collected including vascularization, glomeruli with their capsules, small urinal tubes and small urinary collecting ducts. The BNE will be maintained in survival by connecting its main arteries and veins to an oxygenated artificial circulation of blood plasma or compatible whole blood. Visual observation of this BNE in function makes it possible to detect the different diseased cell segments that will be sampled and replaced, by in vitro microsurgery by rejuvenated and geometrically reconstituted identical cell segments. After checking the good histological and functional integration of the new cell segments on the BNE, this part of the kidney (which may even be a whole kidney) will be reimplanted into the patient with resumption of the vascular and urinary connections. A particularly advantageous biomedical application for this method of cell regeneration generally concerns degenerative diseases of the joints (osteoarthritis). Cartilage chondrocytes, which often degenerate with age, can be removed by biopsy, endoscopy, local surgery, or arthroscopy. separated from their surrounding cartilage. Their core can then be subjected to the regeneration process according to the invention. After multiplication, the reimplantation of the regenerated cells in the original joint should preferably be done in the vicinity, but outside the surfaces of the mobile articular cavity that supports the mechanical loads in order to avoid any crevices in its moving surfaces. . Sometimes a defect of indirect blood supply of chondrocytes, which is mainly by imbibition, must be corrected. We can then consider the graft around or penetrating into the peripheral cartilage with respect to the articular cavity, an autologous vascular functional tissue comprising small arteries - arterioles - capillaries - venules - and small veins, these vascular functional units can advantageously come from a post-regeneration autologous cell culture according to the invention. This implantation of rejuvenated cells may be in the form of preformed three-dimensional lamellae layers according to the local geometry of the previously measured articular cavity, or in the form of a swarming in order to cause the durable emission in particular of signaling proteins. The post-regeneration chondrocytes will progressively reconstitute a thicker, smooth, well lubricated cartilage. Osteoporosis is a degenerative disease of bone tissue that occurs with age. To fight against this disease, it is advisable to regenerate autologous osteoblasts (and possibly osteocytes) and to re-implant them, preferably, at several levels of the bone. Osteoblasts are preferably propagated in culture with artificial geometric solicitations, in particular by imposing mechanical stresses, for example using a support frame. This support frame may comprise at least one mobile side for movements in a plane. Advantageously, two moving sides are used in the culture support frame and / or the possibility of performing motor rotations. dimensional. At the femoral neck, for example, it is possible, under local anesthesia, to easily perform spaced biopsies by introducing a trocar through the trochanteric solid of the femur which is close to the skin. The treatment of osteoblasts and local osteocytes thus collected following the cell regeneration process and the reimplantation of these rejuvenated cells, for example, by the same transtrochanteric route, may allow the local creation of bone remodeling, which thus performs a fundamental reinforcement. bony trabeculae in the direction of the mechanical stresses of the neck and the femoral head upwards as well as downwards towards the body of the femur. In the vertebrae, the main victims of osteoporosis by fractures and flattening, an equivalent process of regeneration of the cells to that of the femur can be implemented, possibly in association with fixers and artificial joints developed by the inventor in the patents. US-6,835,207 and US-6,692,495. Indeed, the main or aggravating cause of osteoporosis is aging, and the treatment of the invention is, here too, a preferred solution. In the spine, settlements or fractures make walking and lower extremity movement difficult. In this case, it is appropriate to take, for example by posterior transcutaneous puncture, in particular some osteoblasts and osteocytes at the level of the main vertebrae affected, to subject them to a treatment according to the invention and to re-implant them, preferably in the vertebra of origin, in the place as close as possible to the origin of the cell to be treated, preferably with prior amplification in vitro. At the level of the long bones one can proceed in an equivalent way.

The present invention can also be applied to subjects who have undergone serious inflammation, in particular by reaction weakenings of the various antibody-producing lymphocytes and pro-cytokines. anti-inflammatories. The regeneration method according to the invention can then allow to revive these lymphocytes in number and function. To do this, these lymphocytes can be subjected to the method according to the invention by placing a lymphocyte nucleus in an oocyte in the presence or absence of traces of antigens created by the infection in question in the oocyte cytoplasm. In the presence of traces of antigens created by the infection placed in the oocyte cytoplasm, the lymphocytes will be rejuvenated and multiplied and then reimplanted into the organism where they will have already "memorized" the dangerous antigens and produce or cause to be made to produce the corresponding antibodies. in large quantity. In the case where antigens are placed in the cytoplasm of CRG, the presence of specific antigens during the cell regeneration process may allow to "memorize" or exteriorize the antigens on the cell membranes and to optimize the reaction of production of the antigens. antibodies by their immediate appearance as rejuvenated lymphocyte functions recur.

The present invention also applies to cancer control. In order to improve traditional anti-cancer treatments that do not take into account individual biological reactions, the treatment of the invention makes it possible to create a method of personalizing cancer control. It is possible to proceed in the following manner: hematopoietic, lymphocyte, dendritic cells are collected at the level of the bone marrow or at the periphery, and different categories are isolated which are subjected to the treatment of the invention. After amplification of these cells in vitro, they are cultured in a nutrient bath in the vicinity of cancer cells taken from the tumor of the patient. It may then be useful to limit the nutrients and oxygen in the culture bath to stimulate competition and survival struggle between the two categories of cells. The genetically rejuvenated lymphocytes of the patient will naturally develop specific antibodies against the antigens cancer cells and against certain substances and biological factors necessary for the metabolism and secretion of cancer cells. It is for example anti-sense or guide RNAs, often small, previously transfected in the DNA, in particular lymphocyte, by plasmids carrying the genes selected or constructed for this purpose. If the lymphocytes succeed in destroying the cancerous cells, they can be reinjected, preferably after their multiplication, into the organism of the patient from which they come. If, on the other hand, the lymphocyte cells fail in the destruction of the cancerous cells, the former must be reinforced, in particular by the injection of plasmids and / or cosmids and / or selected synthetic vectors. These make it possible, for example, to provide DNA polymerases or DNA segments comprising synthetic or natural genes producing new antibodies or specific toxic substances against the cells to be combated. A greater capacity is thus provided to produce antibodies and / or to stimulate lymphocyte metabolisms and mitoses, either by the selection of preferably highly immunogenic cells, such as so-called "memory effectors" with enhanced anti-tumor potential. For example by strengthening the genetic rejuvenation treatment of lymphocytes using the treatment of the invention.

Since cancer cells are autologous, genotypic and differential epigenotypic examination reveals the small part of the genome of cancer cells that differs from autologous normal cells of the same tissue (PGD). The identification of PGD relative to known PGDs from other cancer cells, preferably from the same tissue of other people, allows classification for therapeutic purposes. However equal to PGD the genes involved can produce different _m RNAs including editing (editing) or differential splicing. It is therefore necessary to know the biological and biochemical behaviors of the specific PGD each patient may, in part, vary in response to a therapy, for example biological, of the type of the present invention. In vitro, one can then try to find known innocuous viruses or bacteria such as certain bacteriophages and colifacts selected and / or genetically manipulated.

If an adult homologous foreign cytoplasm (ECH) is introduced into an enucleated oocyte, genetic reprogramming of the mitochondrial DNAs and ribosomal RNAs of the CEH is possible. This possibility can be used to protect an adult nucleus placed in an active oocyte against subsequent transfections by the oocyte cytoplasm as they occur during conventional cloning. Partial cloning removes the nucleus to be treated (NT) from the oocyte before its first cell division and places this reprogrammed nucleus in a cell preferably enucleated identical to its original cell. Thus, the oocyte cytoplasm is distant from the NT nucleus at the time of division of the nucleus, and this division takes place within an original cytoplasm (CO) of the NT nucleus. It is necessary to reprogram the CO genetically and to increase the quantity. To this end, we can take a second oocyte identical to the first and suck part of its cytoplasm and replace it with a cytoplasm of a cell identical to that of the NT core (CCINT). After a desired time, the CCINT is removed from this oocyte and surrounded by the NT nucleus, which has kept some of its cytoplasm of CO origin, with the, or if necessary, several CCINT reprogrammed and recovered, before introducing the NT nucleus, thus re-packaged in a cell of origin of the NT nucleus, preferably enucleated and emptied of a part of its cytoplasm. If necessary, this cell can be enlarged according to one of the membrane manipulations previously described.

The present invention may advantageously also be applied to ulcers. Chronic ulcers, especially in the legs, often very long before healing, and this healing often leaves a significant cutaneous and subcutaneous sequelae. Other ulcers never heal. In this case, it is appropriate to treat by the regeneration method according to the invention at least one epidermodermal functional unit of the patient preferably taken from the healthy skin close to the ulcer and, after multiplication, to implant it. at the level of the ulcer. Implantation can be done directly at the level of the ulcer when a sufficient local blood supply exists without serious infection, or in the negative, to be performed around the ulcer in the healthy cutaneous region. In order to achieve, for example, such an epidermodermic functional unit in simultaneous reprogramming, the CRG (s) intended to accommodate this unit can be quite bulky and can therefore be for example artificially enlarged by the method previously described. At the epidermal level, the cell may for example be chosen from a keratinocyte, Langerhans, Merkel and / or melanocyte cell, taken alone or in combination, while at the dermal level, cutaneous fibroblasts may be taken. The cells of the epidermis and dermis can be placed in separate oocytes. The epidermodermal cells collected after regeneration should as far as possible be positioned and fixed in the culture bath in a reciprocal conformation close to that naturally observed, so as to promote functional cell growth and simplify the implantation of the layer. tissue regenerated on the receiving skin. When culturing the regenerated cells, it may be possible to rearrange the respective position of the different categories of cells, or even to cultivate several variants of assembly intended for grafts at different locations or having a different morphology or function.

It is also known that, with age, DNA copying defects become more important and that the natural mechanisms for repairing the DNA become more important. these defects become less effective. In order to overcome this insufficiency, a device of the invention can provide for locally removing the epidermis and / or the damaged dermis, treating it with the regeneration according to the invention, and then manufacturing from this regenerated tissue. genetically an extract of these cells, which may be fixed for example in a cream, solution or the like for an external cutaneous application. This extract can also be used to create a solution for injection in subcutaneous, intradermal or intra epidermal. It thus becomes possible to quickly and temporarily restore the repair functions of the epidermal and / or dermal DNA. Moreover, in an epidermal application, the invention makes it possible to fight genetically against the senescence of the skin, by modifying the collagens, in particular by rejuvenating them, to restore elasticity to the skin.

Regeneration of necrotic, fibrotic or inactive tissue areas is also an application of the present invention. Such damaged tissue areas appear, for example, at the level of a myocardium, particularly following an infarction or heart failure, or for example at a body that has developed a tumor targeted by a destructive anticancer treatment. In a myocardial application, it may be necessary to control the electrical and mechanical refractory phases of the cells contracting in a synchronous manner in order to avoid cardiac rhythm disturbances during their implantation in the recipient heart. The invention also applies to heart valves. These can be organic, with a limited lifespan (around 15 years). They can also be artificial, with a longer lifespan

(about 30 years), but in this case the subject is subjected to a therapy of anticoagulant for life, very constraining. The invention makes it possible to create a biological, artificial, mixed, or repaired cardiac valve, and to coat the surface of this substrate which is in contact with the blood of the patient. less a regenerated autologous cell layer. This coating can be made from a treatment of cardiovascular endothelial autologous cells previously removed by cardiac or vascular catheterization, treated according to the present invention, and then implanted on the valve. In the case of a plasty, this implantation can be done intraoperatively, that is to say during the operation, covering at least a portion of the valve and the valve ring. In this way, anticoagulant therapy may become unnecessary. The present invention also makes it possible to perform cardiac autografting instead of homologous grafts and mechanical artificial hearts with pneumatic or electrical transcutaneous supply. The inventor was the first in the fifties to implant a totally artificial heart in a dog, allowing him to survive a short time while his natural heart was in a jar (R. Monod, F. Zacouto, E. Coraboeuf and R. Saumont: "Cardiopulmonary bypass allowing the temporary exclusion of the heart and its replacement by an intrathoracic mechanical device", Compt Rend Soc Biol., 150, No. l, 48 (1956)).

In the case of such an autograft, 3D echocardiography can advantageously be used to accurately reconstruct the geometries of the heart in order to best adapt to the thoracic volume specific to each patient (N. Mirochnik, A. Hagège, F. Zacouto and C. Guérot, "Physical reproduction of cardiac structures: a new avenue of exploration in cardiology." Heart and Vessel Diseases Archives, Volume 93, No. 10, October 2000, pp. 1203-1209). The present invention can also assist in the determination of the mechanism responsible for a disorder of the health status of a mammal. Indeed, the root cause of an impairment of the vital balance is sometimes difficult to find. It is then possible to perform a cell regeneration of at least one cell of the suspect tissue and if the resulting reprogrammed tissues differ from normal tissue in its intracellular composition or in its secretions of proteins and peptides in a critical or specific manner the intrinsic causal responsibility of this tissue is demonstrable. By way of example, certain diabetics whose pathology is old may be cited as a cellular regeneration of a pancreatic

Langerhans, taken for example by endoscope, will show an induced secretion of insulin or normal glucagon in contrast to the equivalent cells not subject to cell regeneration. The origin of disease states occurring after a certain age is likely to be revealed by the functional comparison of existing or present suspicious tissue with respect to its now genetically rejuvenated tissue ancestor at a given biological age.

The present invention is also useful for diseases characterized by cell deficiency. In particular, the present invention may allow regeneration and multiplication of β and α cells of islets of

Langerhans that can be re-implanted in the pancreas or elsewhere in order to restore the secretion of insulin or glucagon in an organism. In some cases of destruction of liver tissue (such as cancer, intoxication or cirrhosis), the implantation of regenerated hepatocytes can ensure healing of liver disorders.

Still another application of the present invention is, for example, the maintenance of an implant in a bone using a simple envelope or framework or support of regenerated cells. This application comprises regenerating bone cells, in particular osteoblasts, preferably taken at an early stage of their spontaneous or induced mitosis in a CRG and then placing these regenerated osteoblasts before the end of their mitosis in a receptor cell preferably of osteoblasts, to cultivate osteoblasts in a suitable culture medium in order to obtain appropriate numbers and mechanical behavior and then to distribute the osteoblasts in the form of a sleeve, pedestal, framework, or envelope between an artificial bone implant and the bone. The genetically rejuvenated layer of osteoblasts then ensures a good bonding of the bone implant and the bone by the osteoblastic thrust, and thus reinforces the maintenance of the implant in the bone and strengthens the bone structure itself. In addition, the use of such regenerated cells ensures the long-term maintenance of the implant in the bone and can have a lasting, increased and curative efficacy compared to protein creams based on different BMPs (Bone Morphogenic Proteins) usually implemented. Another possible use of the present invention is to ensure good histocompatibility between a donor graft and the immune system of a recipient. To do this, a healthy cell of the graft member of the recipient can be removed and regenerated according to the described process and then transferred to a suitable receptor cell so as to cause the proliferation of these cells. These cells can then be placed around the donor graft so that the recipient's immune system recognizes the critical molecules on the graft surface as self molecules and thus does not generate a strong immune response in the presence of the graft. . The creation of histocompatibility can in particular be carried out in the following ways:

1) Exchange of chromosomes or segments of chromosomes by genetic micromanipulation during a selected phase of mitosis, which is difficult to achieve today because their micromanipulation is not yet sufficiently precise; nanomechanics currently makes it possible to produce instruments on a chromosome scale to perform, for example, punctures, grafts, aspirations, transfers, cuts and rotations; progress at this level is expected and possible in the near future. 2) Selective destruction of a chromosome segment carrying genes responsible for tissue incompatibility, for example during a mitosis phase, for example using a micro laser beam of diameter equal to or less than 1 micron, surrounded by a cylinder of wider radii of visible light for guiding the laser beam by simple microscopic optical control, which can be advantageously robotic.

The present invention is also of particular interest in the field of dental stomatology. The absence of teeth must often be compensated by ceramic, plastic, etc. metal implants. These implants require a sufficient maxillary bone support base to ensure strong fixation of the implant. In case of insufficiency of the volume or the quality of the requested region of the maxillary it is advantageous to take, for example by mouth, some cells of this place of the bone, to subject them to a cellular regeneration and to a multiplication adequate to provide a small local bone graft that will not only provide a solid bone base but may for example, through signaling proteins, local cytokines and molecules regulating cell activity and those of genetic expressions, gradually strengthen the entire maxillary arch. Advantageously, this bone regeneration is combined with the maxillary bone, which can be done by local injections of regenerated cells into, in contact with, or near the bone, a coating of the implant with at least one layer of bone cells. and / or regenerated periodontal, which will improve the fixation, the viscoelastic strength and the corresponding strength of the implant in the bone, as well as the strength of the bone itself.

Another example of application of the cell regeneration method according to the invention relates to fractures and bone surgery. Some bone fractures and malformations require surgery that sometimes requires extra bone mass graftable and solid. This can be achieved by genetic rejuvenation of local cells with multiplication each time the final surgery can be delayed by at least two weeks. This is particularly the case for interventions for pseudarthrosis, vertebral bone deformities of children or degenerative, arthritis or deforming arthrosis. In vitro cell multiplication of osteoblast cells should preferably be carried out taking into account, as soon as they are cultured, the mechanical stresses they must withstand, for example after implantation in the femur, maxillary, vertebrae, etc. In practice, it is a matter of organizing this cell culture if possible physiologically confluent and in a suitable feeder bath, but inside a support frame of which at least one side is movable in a plane, or in two planes simultaneously , which makes a motor rotation possible. The movements and mechanical forces periodically imposed on the growing tissue in its adapted nutrient solution will be of a magnitude, suddenness and progressively increasing strength, but always in a similar main orientation in order to provoke mineral and trabecular structures in the right direction. The lines of force and mechanical strength of these structures, in one or more directions, correspond to the forces, damping and viscoelasticities that the regenerated bone tissue will have to support after implantation. The inventor has developed an original adjustable vertebral fixator (US-6,835,207) and an original adjustable vertebral disc (US-6,692,495), both of which can advantageously be combined with vertebral tissue resulting from such cellular regeneration, and for example serve as a pedestal base for fixation pedicle screws or for filling crowded or fractured vertebrae, or as a supporting framework.

Another application of the invention relates to non-autologous grafts. Indeed, a major problem concerns the rejection of transplants by the recipient. However, it is known that the fetal cells or near embryonic cells are less rejected. Sufficiently rejuvenated cells, for example by several successive treatments according to the invention, would make it possible to reduce the problem of rejections of non-autologous grafts. It should be noted that changing the biological age of a cell

(rejuvenation or aging) can be measured by different methods. Thus, the times or speeds that a cell takes to recover its membrane potential and its action potential after being subjected to stress (such as a lack of oxygen or an excess of potassium) can be compared before and after treatment. If the recovery time is shorter, then the cell is functionally rejuvenated. Other methods consist in comparing, before and after treatment, the repetition rates of the mitoses or the mitoses themselves, the cicatrization rates, or the changes in the dimensions (volume) of the telomeres. It should also be noted that the process according to one aspect of the invention, designated by the term "partial cloning", differs from conventional cloning. Indeed, in conventional cloning, the nucleus of a cell is transferred into an oocyte, will divide and become able to reproduce in utero or artificially in vitro the original tissue of said nucleus. Now, this means that the nucleus is in contact with a cytoplasm containing mitochondria and ribosomes capable of influencing the function and / or evolution of the nucleus, in particular via foreign DNAs and / or RNAs, because oocytes they contain. This means that unless the oocyte is derived from the mother of the original cell soon after delivery, conventional cloning does not produce purely autologous cells and tissues. Moreover, even if the oocyte comes from the mother, it is possible that it has different characteristics due in particular to the influence of the environment, therapies, diseases, age, etc. To obtain a purely autologous tissue, it would be necessary to use an oocyte of the mother obtained at the time of the birth of origin, which is rarely feasible.

On the other hand, in partial cloning, which consists in temporarily introducing a nucleus into an oocyte for a short time, then retransferring it to an autologous recipient cell, preferably identical to its cell of origin, the tissue obtained is purely autologous, whatever the oocyte (or CRG or equivalent MRG cell) used. This can make it possible to use an oocyte of a mammal not necessarily identical while ensuring maximum genetic purity. The methods according to the invention can also be used to obtain embryonic cells from an adult nucleus. For this, one surrounds the previously reprogrammed nucleus preferably with additional cytoplasm autologous to the nucleus, as previously described. Then the treated nucleus thus packaged is placed in an oocyte (or MRG), enlarged if necessary according to the invention, and the embryonic cell divisions are allowed to develop. Thus, embryonic or fetal cells from a younger nucleus are created and the differences in biological age between this nucleus and the embryonic or fetal cells created are reduced.

Although the present invention has been described with reference to various aspects thereof, and to the means of various application examples, it is understood that it is not limited thereto, the scope of the invention being given by the appended claims.

Claims

claims

1. A process for the genetic and epigenetic treatment of cells to be treated, characterized in that it comprises the following steps:

provide at least one cell to be treated,

provide a genetic reprogramming medium (MRG), comprising at least the natural cytoplasm of at least one genetic reprogramming cell (CRG) and / or synthetic cytoplasm, and

contacting at least a portion of at least one nucleus of at least one cell to be treated with said MRG to modify the biological age and / or repair said at least one cell to be treated.

The process of claim 1, further comprising the steps of:

separating from said MRG at least said part of at least one nucleus of the cell to be treated, introducing at least said part of at least one nucleus of the cell to be treated and separated into a receptor cell, preferably emptied of its nucleus , preferably autologous with respect to the nucleus of the cell to be treated, advantageously close to or identical to the cell to be treated, said receptor cell then being able to multiply.

3. A process according to claim 2, wherein said multiplication of autologous cells forms a purely autologous tissue with respect to the organism and / or the tissue from which said nucleus of the cell to be treated is produced, without DNA interference and / or foreign RNA, in particular mitochondrial and / or ribosomal from said MRG.

The method of claim 2 or 3, wherein said step of contacting at least a portion of at least one core of at least one cell to be treated with said MRG comprises taking and transferring at least said portion of said at least one nucleus from said at least one cell to be treated in said MRG.

5. A method according to any one of claims 2 to 4, wherein said step of separating said MRG at least the nucleus of the cell to be treated is performed before the end of the first mitosis of said nucleus.

6. A method according to any one of claims 2 to 4, wherein said step of separating said MRG at least the nucleus of the cell to be treated is performed after one or more mitoses of said nucleus.

7. A process according to any one of claims 2 to 6, wherein said at least a portion of at least one nucleus of at least one cell to be treated comprises at least one chromosome.

8. A process according to any one of claims 2 to 7, wherein the biological age of said at least one cell to be treated is changed.

9. The method of claim 8, wherein said at least one cell to be treated is rejuvenated.

10. The method of claim 9, wherein said at least one cell to be treated is rejuvenated several times successively, from so that his biological age is brought back to a very young age, in particular fœtal or near embryonic.

11. The method of claim 9 or 10, wherein after rejuvenation of the biological age of a cell, the rejuvenated nucleus is removed and replaced by a nucleus of the non-rejuvenated tissue of origin, so that said non-rejuvenated nucleus is in contact with a rejuvenated cytoplasm, the interaction between them causing a rejuvenation of said non-rejuvenated nucleus.

12. The method of claim 8, wherein said at least one cell to be treated is aged.

13. The method of claim 12, wherein is removed at least a portion of a nucleus of a young cell, including fetal or embryonic, diseased, such as a chromosome, and replaced by at least an equivalent portion an older healthy nucleus of the same tissue, preferably HLA compatible, the interaction between the young cytoplasm with at least the part of the older nucleus promoting a temporary acceleration of aging of at least the cytoplasm of the young cell.

14. The method of claim 13, wherein the aging is performed by multiplication of cells in a cell culture.

15.- Method according to claim 13 or 14, wherein, during the deployment of chromosomes preferably in metaphase, at least a portion of a diseased chromosome is destroyed, at least a portion equivalent of an equivalent chromosome of an older healthy cell, preferably HLA compatible, being removed and implanted in the diseased nucleus.

16. The method of claim 15, wherein the destruction of at least a portion of a diseased chromosome is performed with an ultrathin laser beam, preferably of diameter equal to or less than 1 micron.

17. A method according to any one of claims 8 to 16, wherein the modification of the biological age of a cell is measurable by comparing before and after treatment of the speed that a cell has to recover its membrane potential. and its action potential after being stressed, such as a lack of oxygen or an excess of potassium, and / or by comparison before and after treatment of the repetition rate of mitoses or mitoses themselves , and / or by comparison before and after treatment of the cicatrization rate and / or by comparison before and after treatment of the telomere volume.

18. A method according to any one of claims 2 to 9, wherein said at least one cell to be treated is repaired, especially in its chromosomal constitution.

19. The method of claim 18, wherein a portion of a chromosome to be treated is removed, in particular by means of an ultrathin laser beam, then an equivalent healthy part of an equivalent chromosome is taken, in particular by laser cutting. , and reintroduced into the chromosome to be treated, in particular by means of plasmids, phagemids, synthetic vectors, or micromanipulations, for repairing said chromosome to be treated.

20. The method of claim 19, wherein said deleted portion of the chromosome to be treated is the part whose genes are responsible for graft rejection.

21. A method according to any one of claims 2 to 20, wherein said MRG further comprises substances capable of activating nuclear metabolism, such as cells or cell extracts appearing during healing and / or proteins or peptides for signaling or stimulating metabolism and / or growth factors and / or cells or extracts of cancer cells.

22. The method as claimed in claim 2, in which at least one cell to be treated is subjected to temporary treatment with selected cancer cells, intracellularly or extracellularly.

The method of any one of claims 2 to 22, comprising the steps of:

to temporarily transfer cytoplasm from a cell identical to the cell to be treated in an MRG; to separate said transferred cytoplasm from said MRG after reprogramming it;

- Surrounding said nucleus of the cell to be treated separated from said MRG with said reprogrammed cytoplasm before the introduction of the assembly into the recipient cell.

24.- A method of treatment against cancer, characterized in that it comprises the following steps:

- take at least one hematopoietic cell from the bone marrow or from its periphery,

provide a genetic reprogramming medium (MRG) comprising at least the natural cytoplasm of at least one genetic reprogramming cell (CRG) and / or synthetic cytoplasm,

contacting at least a portion of a nucleus of a hematopoietic cell taken with said MRG,

separating said MRG from at least said part of a nucleus of a sampled hematopoietic cell,

- Introducing at least said part of a nucleus of a hematopoietic cell thus separated in a hematopoietic cell receptor, preferably emptied of its nucleus, preferably autologous with respect to the nucleus of the hematopoietic cell, advantageously close or identical, to genetically reprogram and / or rejuvenate said receptor hematopoietic cell,

to multiply in vitro said reprogrammed and / or rejuvenated receptor cell,

reinjecting said reprogrammed and / or rejuvenated cells to fight cancer.

25. The method as claimed in claim 24, in which said reprogrammed and / or rejuvenated hematopoietic cells are, before reinjection, brought into contact with cancer cells taken from the tumor in vitro, in particular so that said hematopoietic cells are reprogrammed and / or rejuvenated develop specific antibodies against cancer cell antigens.

The method according to claim 25, wherein, before reinjection, said reprogrammed and / or rejuvenated hematopoietic cells are reinforced, for example by injection of plasmids and / or cosmids and / or synthetic vectors, such as copolymer blocks, selected to increase the ability to produce antibodies and / or to stimulate the metabolisms and / or mitoses of said hematopoietic cells and / or to enhance the antibiotic or destructive power against targeted bacteria, fungi or viruses, and / or by selecting immunogenic preferable cells, such as T cells, and / or enhancing the genetic rejuvenation of said reprogrammed and / or rejuvenated hematopoietic cells.

27.- System for the genetic and epigenetic treatment of cells to be treated, characterized in that it comprises:

at least one cell to be treated,

a genetic reprogramming medium (MRG), comprising at least the natural cytoplasm of at least one genetic reprogramming cell (CRG) and / or synthetic cytoplasm, and means for contacting at least one part of at least one a nucleus of at least one cell to be treated with said MRG to modify the biological age and / or repair said at least one cell to be treated.

28. The system according to claim 27, wherein said system is a system for genetic regeneration of differentiated cells, in particular for their genetic rejuvenation, said at least one cell to be treated being a differentiated cell, and said medium of genetic reprogramming (MRG) comprising at least the cytoplasm of at least one oocyte, embryonic, fetal or cancer cell, and / or synthetic cytoplasm.

The system of claim 27 or 28, further comprising:

means for separating said MRG from at least the nucleus of the cell to be treated before the end of its first mitosis,

means for introducing the nucleus of the cell to be treated thus separated into a receptor cell, preferably emptied of its nucleus, preferably autologous with respect to the nucleus of the cell to be treated, advantageously close to or identical to the cell to be treated, said receiving cell then being a genetically rejuvenated cell able to multiply.

The system of claim 29, wherein said means for contacting at least the nucleus of a cell to be treated with said MRG comprises means for taking and transferring at least said nucleus from said cell to be treated in said MRG.

31. The system according to claim 30, wherein said means for taking and transferring at least said nucleus into said MRG are adapted to collect and transfer said nucleus of the cell to be treated together with at least a portion of its cytoplasm in order to find in the MRG some cytoplasmic components initially present in the differentiated cell, such as the endoplasmic reticulum, the golgy apparatus, the ribosomes and / or the mitochondria.

The system of claim 30 or 31, wherein the nucleus of the cell to be treated for transfer into the MRG is removed at an early stage of mitosis, such as prophase, pro-metaphase or metaphase.

The system according to any one of claims 30 to 32, wherein the nucleus of the cell to be treated transferred into the MRG is separated from the MRG at the metaphase, anaphase or telophase stage.

The system of claim 27 or 28, wherein said means for contacting at least the nucleus of a cell to be treated with said MRG comprises means for transferring MRG into the cell to be treated.

The system of claim 34, wherein said means for transferring MRG into a cell to be treated comprises means for creating at least one slit or aperture in the membrane of said cell to be treated, and means, such as a pipette, for transferring MRG into said cell to be treated through said at least one slot or opening.

The system of claim 34 or 35, wherein said means for transferring MRG into a cell to be treated comprises means for at least partially separating said MRG from said cell to be treated.

37. The system of claim 34, 35 or 36, comprising means for permanently closing the cell to be treated with at least a portion of the remaining MRG included in it.

The system of claim 36 or 37, wherein said means for transferring MRG into a cell to be treated comprises means for opening the membrane of an embryonic or fetal egg cell, means for opening the membrane of an cell to be treated, means for bringing said openings in contact, means for compressing said oocyte, embryonic or fetal cell to at least partially transfer its cytoplasm into the cell to be treated.

39. The system of claim 38, wherein said means for transferring MRG into a cell to be treated further comprises means for at least partially returning said transferred cytoplasm to its original cell after a predetermined time, in particular by decompression of said oocyte, embryonic or fetal cell.

40. System according to any one of claims 27 to 39, wherein said MRG comprises at least the cytoplasmic extract of at least one oocyte, embryonic or fetal cell, said extract being obtained by treatment of the cytoplasm.

41. System according to any one of claims 27 to 40, wherein said MRG comprises at least one genetic reprogramming cell (CRG), which can be a cell embryonic, oocyte or fetal, the nucleus of which has preferably been extracted.

The system of any one of claims 27 to 41, wherein said MRG further comprises substances capable of activating nuclear metabolism, such as cells or cell extracts occurring during healing and / or signaling proteins and / or growth and stimulation factors.

43. System according to any one of claims 27 to 42, comprising means for injection and / or implantation, such as by endoscopy, treated cells, able to distribute said cells within, near or remotely. of a tissue to be treated.

44. System according to any one of claims 27 to 43, wherein the system is suitable for the treatment of cells to be treated such as cardiac, renal, bone, dental, periodontal, cartilaginous, pancreatic, hepatic, nerve, prostatic cells. , hematopoietic, immune, pulmonary, arterial, retinal, cutaneous, dermal, epidermal, glandular, tendinous, vascular, splenic, parathyroid, adrenal and / or digestive and respiratory tract.

A system according to any one of claims 27 to 44, wherein a plurality of cells representing an organic functional unit are treated together and / or reassembled after isolated genetic treatment to form a genetically treated organic functional unit, including rejuvenated .

46. The system for genetic regeneration of differentiated cells according to claim 28, comprising:

at least one differentiated cell,

cell reprogramming means, comprising at least one genetic reprogramming cell (CRG), formed in particular of an oocyte or of an embryonic cell,

means for suppressing, preferably completely, the nucleus of the at least one CRG,

means for taking and transferring the nucleus of a differentiated cell into a respective CRG,

means for extracting the CRG, the nucleus transferred from the differentiated cell before the end of its first mitosis,

means for introducing the nucleus of the differentiated cell extracted into a differentiated receptor cell, preferably emptied of its nucleus, preferably autologous with respect to the nucleus of the differentiated cell, advantageously close to or identical to the differentiated cell, said receptor cell being then a regenerated cell capable of multiplying, the resulting tissue being intended to repair and / or replace and / or be positioned in a tissue to be regenerated.

The system of claim 46, further comprising:

means for taking at least one determined chromosome on the original nucleus of the CRG extracted from said CRG,

means for implanting this or these chromosome (s) in the nucleus of the differentiated cell transferred into and / or extracted from said

MAF.

48. The system of claim 47, further comprising means for removing from the nucleus of the transferred differentiated cell. in said CRG, the chromosome (s) corresponding to the chromosome (s) taken from the original nucleus of CRG.

49. System according to any one of claims 27 to 48, comprising means for in vitro cell multiplication of treated cells, comprising a support frame receiving a nutrient bath containing said cells treated in culture, said support frame being subjected to movements. and / or mechanical forces during said multiplication.

50. System according to claim 49, wherein said support frame comprises at least one movable side in a plane, preferably at least two moving sides in two different planes and in particular adapted to perform a three-dimensional driving rotation.

51. System according to any one of claims 27 to 50, comprising mechanical and / or biological support means for the in vitro growth of the treated cells, such as an implant having an enlarged non-smooth surface adapted for this purpose. support means being able to be positioned in the tissue to be treated, said treated cells then forming a cellular cement between said tissue to be treated and said support means.

The system of claim 51, wherein said growth support means of the treated cells comprises an artificial or biological bone implant.

53. A method of genetic and epigenetic treatment, comprising the use of at least one genetic reprogramming cell and at least one differentiated cell for the manufacture of at least one regenerated differentiated cell capable of being reimplanted to treat various pathologies, diseases, and / or tissue insufficiencies, comprising the following steps: - providing at least one genetic reprogramming cell

(CRG), consisting of an oocyte or an embryonic cell,

- provide at least one differentiated cell,

- delete, preferably completely, the core of the CRG,

- to collect and transfer the nucleus of a differentiated cell in CRG,

extracting the nucleus of the differentiated cell transferred into CRG, before the end of its first mitosis,

introducing the nucleus of the differentiated cell extracted into a differentiated cell, preferably emptied of its nucleus, preferably autologous with respect to the nucleus of the differentiated cell, advantageously close to or identical to the differentiated cell, so as to form a regenerated differentiated cell ,

- Multiply said regenerated differentiated cell.

54. The method according to claim 53, comprising the use of at least one genetic reprogramming cell and at least one chondrocyte for the production of implantable regenerated chondrocytes, preferably by endoscopy, ultrasound-controlled transcutaneous puncture. , or arthroscopy, in bone or joint cartilage prepared for this purpose, in particular outside its articular surface, to treat osteoarthritis by regenerating said cartilage.

The method of claim 53, comprising using at least one genetic reprogramming cell and at least one a cardiac cell for the manufacture of regenerated cardiac tissue capable of being implanted in the myocardial tissue to regenerate an injured area, in particular following a myocardial infarction or a heart failure.

56. The method of claim 53, comprising the use of at least one genetic reprogramming cell and at least one healthy cell derived from a tissue to be subjected to anticancer treatment, for the manufacture of healthy regenerated cells capable of to be implanted in the tissue targeted by the anti-cancer treatment to regenerate said tissue after the end of the anti-cancer treatment.

57. The method as claimed in claim 53, comprising the use of at least one genetic reprogramming cell and at least one Langerhans β and / or α cell for the manufacture of regenerated cells capable of being implanted in the body. , in particular subcutaneously, and / or in the pancreas, in order to reconstitute a stock of rejuvenated β and / or α cells with normalized endocrine function, and to increase the amount of insulin and / or glucagon produced, to regenerate the function of secretion of insulin and / or glucagon.

58. The method according to claim 53, comprising the use of at least one genetic reprogramming cell and at least one epidermal-dermal unit of rejuvenated autologous tissue, in particular for the manufacture of functional regenerated epidermal-dermal units capable of to be implanted in or near an ulcerous tissue for the treatment of an ulcer or any irreversible lesion or skin abnormality.

59. The method of claim 53, comprising the use of at least one genetic reprogramming cell and at least one osteoblast for the manufacture of regenerated osteoblasts able to be implanted in a bone osteoporosis victim to regenerate said bone.

60. The method of claim 53, comprising the use of at least one genetic reprogramming cell and at least one bone cell for the manufacture of regenerated bone cells adapted to be implanted in a bone receiving an implant to promote the securing said implant in said bone, forming during their growth a cellular cement, solidarisant the bone and the implant.

61. The method of claim 53, comprising the use of at least one genetic reprogramming cell and at least one epidermal and / or dermal cell for the production of regenerated epidermal and / or dermal cell extract for treating the damaged epidermis and / or dermis and restore its repair functions of epidermal and / or dermal DNA.

The method of claim 61, wherein the cell extract is fixed in a cream for external skin application.

The method of claim 61, wherein the cell extract is in the form of a solution for injection subcutaneously, intradermally or intra epidermally.

The method according to any of claims 53 to 63, wherein said in vitro cell multiplication of the regenerated cells is carried out by means of a support frame receiving a bath feeder containing said regenerated cells in culture, said support frame being subjected to movements and / or mechanical forces during said multiplication.

65.- A method according to claim 64, wherein said support frame comprises at least one movable side in a plane, preferably at least two moving sides in two different planes and in particular adapted to perform a three-dimensional driving rotation.

66. The use of a system and / or method according to any of the preceding claims, for at least partially coating a biological, artificial or repaired cardiac valve of at least one autologous regenerated cell layer.

67. Use of a system and / or a method according to any one of claims 1 to 65, for regenerating at least partially the part of a maxillary bone and the tissues of the periodontal intended to receive a dental implant.

68.- Use of a system and / or a method according to any one of claims 1 to 65, for regenerating at least partially the epidermis by regeneration of fibroblasts, causing a modification of collagens.